Received on August 19, 2005
Accepted on November 28, 2005

Drug Monitoring and Toxicology

Measurement of Erythrocyte ITPA Activity by HPLC and Correlation of ITPA Genotype-Phenotype in a Caucasian Population

¹ Central Institute of Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Stuttgart, Germany
² Department of Clinical Chemistry, Georg-August-University, Göttingen, Germany

Methods: The enzymatic conversion of ITP to inosine monophosphate (IMP) was terminated by perchloric acid and saturated dipotassium hydrogenphosphate. We quantified the product IMP at 262 nm after separation on an aqua perfect C18 column, using 20 mmol/L, pH 2.5 phosphate buffer. We also genotyped samples for ITPA 94C>A and IVS2 + 21A>C using real-time fluorescence PCR.

Results: The assay was linear to 3 mmol/L IMP (500 µmol/(g Hb · h)) with an LLQ of 4 µmol/L (0.5 µmol/(g Hb · h)). With IMP-enriched samples, imprecision was 3.6% within a day and 4.9% from one day to the next, and the inaccuracy was 5.2%. Imprecision using pooled erythrocytes was 3.8% within a day and 7.5% from one day to the next. ITPA activity in 130 healthy controls ranged between <0.5 and 408 µmol IMP/(g Hb · h). Mutant allele frequencies were 0.062 (94C>A) and 0.131 (IVS2 + 21A>C). Using a cutoff at 125 µmol, IMP/(g Hb · h) phenotyping detected all 94C>A mutant cases, all 94C>A and IVS2 + 21A>C compound heterozygotes, all IVS2 + 21A>C homozygotes, and 6 of 24 IVS2 + 21A>C heterozygote only cases. A novel IVS2 + 68T>C mutation was found.

Conclusion: The HPLC procedure provided an excellent ITPA phenotype-genotype correlation and led to the discovery of a novel IVS2 + 68T>C mutation. The method should facilitate investigation of the role of ITPA activity for drug toxicity during thiopurine therapy.